Amplification of nucleic acids

ABSTRACT

Provided herein is technology relating to amplification of nucleic acids and particularly, but not exclusively, to compositions and methods for doing improving the polymerase chain reaction and providing reagents for polymerase chain reaction with improved stability.

This application is a continuation of U.S. patent application Ser. No.16/722,239, filed Dec. 20, 2019, which claims priority to U.S.provisional patent application Ser. No. 62/786,860, filed Dec. 31, 2018,which is incorporated herein by reference in its entirety.

FIELD

Provided herein is technology relating to amplification of nucleic acidsand particularly, but not exclusively, to compositions and methods forconducting polymerase chain reactions and providing reagents forpolymerase chain reactions with improved stability.

BACKGROUND

Many in vitro diagnostic assays based on the polymerase chain reaction(PCR) rely on fast polymerase enzymes. Accordingly, fast PCR enzymeshave been engineered to deliver increased speed and processivity, butmay also exhibit an increased propensity to generate spuriousamplification products from non-specific priming and/or dimer-primerformation. The formation of these non-specific products can negativelyimpact the overall efficiency of PCR and/or RT-PCR by interfering withtarget amplification and/or by depleting reaction resources.

Numerous technologies have been developed to improve the amplificationof nucleic acids, e.g., by PCR. For example, compositions and methodsare available that reduce non-specific priming and/or dimer-primerformation in the PCR. One particular strategy comprises use of “hotstart” primers and “hot start” dNTPs that are produced with athermolabile chemical modification that prevents extension prior toincubation at an elevated temperature at the beginning of a thermalcycling program. However, “hot start” mechanisms can negatively impactRT-PCR because most reverse transcriptase enzymes are irreversiblyinactivated by the elevated temperature of the “hot start” thermalcycling step. Other PCR and RT-PCR enhancers include betaine,tetramethylammonium chloride (TMAC), formamide, and dimethyl sulfoxide(DMSO), which generally function by increasing the specificity ofhybridization and increasing the Tm of primers/probes.

Although non-specific priming and/or dimer-primer formation can occurduring reverse transcription and/or PCR steps, it can also occur duringmaster mix preparation and during reaction setup. These problems arechallenges to manufacturers of PCR and RT-PCR assays, especially whenmanufacturing steps require assembly of complete master mixes, e.g.,comprising enzymes, activation reagent, primers, and/or probes) as asingle bulk reagent. The long lead times typically associated withpreparing, mixing, and filling such bulk reagents can increase thelikelihood non-specific priming and/or dimer-primer formation duringmanufacturing and, thereby, lead to a decline in assay performance.

SUMMARY

The technology provided herein relates to use of2-methyl-4-isothiazolin-3-one (MIT) as an enhancer of PCR. MIT has beenused as a biocide (e.g., in the preservative sold under the trade nameof PROCLIN 950).The technology provided herein comprises use of MIT asan enhancer of the PCR and to provide increased stability of activatedPCR and/or RT-PCR master mixes (e.g., during master mix reagentmanufacturing). During the development of embodiments of the technologyprovided herein, experiments were conducted to test the use of MIT inPCR to disrupt and/or preventing non-specific priming and/orprimer-dimer formation. Data collected during these experimentsdemonstrated that inclusion of MIT in PCR/RT-PCR reactions preventsand/or disrupts non-specific priming and/or primer-dimer formation,thereby reducing the accumulation of non-specific product (see, e.g.,Example 1 and Example 2). Further, these data indicated that MITprovides a general enhancement of PCR/RT-PCR assay performance (see,e.g., Example 3 and Example 4) and improved stability of activatedmaster mix (see, e.g., Example 5).

Accordingly, the technology finds use as a general enhancer in PCRand/or RT-PCR applications; and 2) to improve stability of PCR and/orRT-PCR master mixes (e.g. during master mix reagent manufacturing). Insome embodiments, the technology provides a nucleic acid amplificationcomposition. In some embodiments, the nucleic acid amplificationcomposition comprising a polymerase, one or more primers (e.g., a firstprimer and a second primer), nucleotides, and 0.0001 to 0.1% (w/w)2-methyl-4-isothiazolin-3-one (MIT) (e.g., 0.001 to 0.01% (w/w) MIT)).In some embodiments, the nucleic acid amplification compositioncomprises 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007,0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016,0.0017, 0.0018, 0.0019, 0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025,0.0026, 0.0027, 0.0028, 0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034,0.0035, 0.0036, 0.0037, 0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043,0.0044, 0.0045, 0.0046, 0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052,0.0053, 0.0054, 0.0055, 0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061,0.0062, 0.0063, 0.0064, 0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070,0.0071, 0.0072, 0.0073, 0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079,0.0080, 0.0081, 0.0082, 0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088,0.0089, 0.0090, 0.0091, 0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097,0.0098, 0.0099, 0.0100, 0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106,0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115,0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124,0.0125, 0.0126, 0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133,0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142,0.0143, 0.0144, 0.0145, 0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w)MIT. In some embodiments, the nucleic acid amplification compositionfurther comprises a target template.

In some embodiments, the nucleic acid amplification composition furthercomprises a detectably labeled probe or intercalating dye. Thetechnology is not limited in the detectable label or intercalating dye.For instance, in some embodiments, the detectable label is a fluorescentmoiety, e.g., a dye that can be synthesized or obtained commercially(e.g., from Operon Biotechnologies, Huntsville, Alabama). A large numberof dyes (greater than 50) are available for application in fluorescenceexcitation applications. These dyes include those from the fluorescein,rhodamine, AlexaFluor, Bodipy, Coumarin, and Cyanine dye families.Specific examples of fluorophores include, but are not limited to, FAM,TET, HEX, Cy3, TMR, ROX, VIC (e.g., from Life Technologies), Texas red,LC red 640, Cy5, and LC red 705. In some embodiments, dyes with emissionmaxima from 410 nm (e.g., Cascade Blue) to 775 nm (e.g., Alexa Fluor750) are available and can be used. Of course, one of ordinary skill inthe art will recognize that dyes having emission maxima outside theseranges may be used as well. In some cases, dyes ranging between 500 nmto 700 nm have the advantage of being in the visible spectrum and can bedetected using existing photomultiplier tubes. In some embodiments, thebroad range of available dyes allows selection of dye sets that haveemission wavelengths that are spread across the detection range.Detection systems capable of distinguishing many dyes are known in theart.

In some embodiments, the nucleic acid amplification composition does notcomprise a primer dimer (e.g., comprising said first primer and saidsecond primer) (e.g., a detectable level of primer dimer).

In some embodiments, the technology provides an oligonucleotide reagent.In some embodiments, the oligonucleotide reagent comprises one or moreprimers (e.g., a first primer and a second primer), a detectably labeledprobe, nucleotides, and 0.0001 to 0.1% (w/w) MIT. In some embodiments,the oligonucleotide reagent comprises a first primer, a second primer, adetectably labeled probe, nucleotides, and 0.001 to 0.01% (w/w) MIT. Insome embodiments, the oligonucleotide reagent comprises a first primer,a second primer, a detectably labeled probe, nucleotides, and 0.0001,0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010,0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019,0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028,0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037,0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046,0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055,0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064,0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073,0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082,0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091,0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100,0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109,0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118,0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127,0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136,0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145,0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT. In someembodiments, the oligonucleotide reagent further comprises a seconddetectably labeled probe (e.g., comprising a fluorescent moiety asdescribed above). In some embodiments, the oligonucleotide reagentfurther comprises a reference dye (e.g., a fluorescent dye as describedabove). In some embodiments, the oligonucleotide reagent furthercomprises a third primer and/or a fourth primer.

In some embodiments, the technology provides kits (e.g., kits fordetecting a nucleic acid by realtime PCR (e.g., RT-PCR)). For example,in some embodiments, kits are provided herein that comprise anoligonucleotide reagent comprising a one or more primers (e.g., firstprimer and a second primer), a detectably labeled probe, nucleotides,and 0.0001 to 0.1% (w/w) MIT; and an activation reagent comprisingmanganese chloride and 0.0001 to 0.1% (w/w) MIT. In some embodiments,kits further comprise a thermostable polymerase in a buffered solution.In some embodiments, the thermostable polymerase is rTth polymerase.However, the technology is not limited to this particular polymerase andcomprises embodiments comprising other polymerases known in the art(e.g., as described herein). In some embodiments, kits further comprisean internal control comprising a control nucleic acid and 0.0001 to 0.1%(w/w) MIT.

The technology relates to providing shelf-stable reagents (e.g.,reagents for realtime PCR). In some embodiments, the technology providesa commercial scale mastermix composition having a volume greater than 10mL (e.g., greater than 100 mL, greater than 1 liter) and comprising afirst primer, a second primer, a detectably labeled probe, nucleotides,and 0.0001 to 0.1% (w/w) MIT (e.g., 0.001 to 0.01% (w/w) MIT). In someembodiments, the commercial scale mastermix composition comprises afirst primer, a second primer, a detectably labeled probe, nucleotides,and 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008,0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017,0.0018, 0.0019, 0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026,0.0027, 0.0028, 0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035,0.0036, 0.0037, 0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044,0.0045, 0.0046, 0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053,0.0054, 0.0055, 0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062,0.0063, 0.0064, 0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071,0.0072, 0.0073, 0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080,0.0081, 0.0082, 0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089,0.0090, 0.0091, 0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098,0.0099, 0.0100, 0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107,0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116,0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125,0.0126, 0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134,0.0135, 0.0136, 0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143,0.0144, 0.0145, 0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT.

In some embodiments, the technology provides a commercial scalemastermix composition having a volume greater than 10 mL (e.g., greaterthan 100 mL, greater than 1 liter) and comprising manganese chloride and0.0001 to 0.1% (w/w) MIT. (e.g., 0.001 to 0.01% (w/w) MIT). In someembodiments, the commercial scale mastermix composition comprisesmanganese chloride and 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006,0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015,0.0016, 0.0017, 0.0018, 0.0019, 0.0020, 0.0021, 0.0022, 0.0023, 0.0024,0.0025, 0.0026, 0.0027, 0.0028, 0.0029, 0.0030, 0.0031, 0.0032, 0.0033,0.0034, 0.0035, 0.0036, 0.0037, 0.0038, 0.0039, 0.0040, 0.0041, 0.0042,0.0043, 0.0044, 0.0045, 0.0046, 0.0047, 0.0048, 0.0049, 0.0050, 0.0051,0.0052, 0.0053, 0.0054, 0.0055, 0.0056, 0.0057, 0.0058, 0.0059, 0.0060,0.0061, 0.0062, 0.0063, 0.0064, 0.0065, 0.0066, 0.0067, 0.0068, 0.0069,0.0070, 0.0071, 0.0072, 0.0073, 0.0074, 0.0075, 0.0076, 0.0077, 0.0078,0.0079, 0.0080, 0.0081, 0.0082, 0.0083, 0.0084, 0.0085, 0.0086, 0.0087,0.0088, 0.0089, 0.0090, 0.0091, 0.0092, 0.0093, 0.0094, 0.0095, 0.0096,0.0097, 0.0098, 0.0099, 0.0100, 0.0101, 0.0102, 0.0103, 0.0104, 0.0105,0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114,0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123,0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132,0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139, 0.0140, 0.0141,0.0142, 0.0143, 0.0144, 0.0145, 0.0146, 0.0147, 0.0148, 0.0149, or0.0150% (w/w) MIT.

In some embodiments, the technology provides methods for detecting anucleic acid (e.g., by realtime PCR). For example, in some embodiments,methods comprise providing a reaction composition comprising apolymerase, a first primer, a second primer, nucleotides, and 0.0001 to0.1% (w/w) MIT (e.g., 0.001 to 0.01% (w/w) MIT). In some embodiments,the methods comprise providing a reaction composition comprising apolymerase, a first primer, a second primer, nucleotides, and 0.0001,0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010,0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019,0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028,0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037,0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046,0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055,0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064,0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073,0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082,0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091,0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100,0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109,0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118,0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127,0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136,0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145,0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT. In someembodiments, methods further comprise adding a sample to said reactioncomposition. In some embodiments, methods further comprise providing adetectably labeled probe (e.g., wherein said label is a fluorescentmoiety as described herein) or an intercalating dye. In someembodiments, methods further comprise thermocycling said reactioncomposition. In some embodiments, methods further comprise measuring thefluorescence emission of said reaction composition.

In some embodiments, methods for detecting a nucleic acid (e.g., byrealtime PCR) comprise storing a realtime PCR reagent comprising 0.0001to 0.1% (w/w) MIT (e.g., 0.001 to 0.01% (w/w) MIT) and at least one of apolymerase, a first primer, a second primer, and/or nucleotides for atleast 1 day, 1 week, or 1 month. In some embodiments, the realtime PCRreagent is used to prepare a reaction composition comprising apolymerase, a first primer, a second primer, nucleotides, and 0.0001 to0.1% (w/w) MIT (e.g., 0.001 to 0.01% (w/w) MIT). In some embodiments,the methods comprise using a realtime PCR reagent to prepare a reactioncomposition a polymerase, a first primer, a second primer, nucleotides,and 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008,0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017,0.0018, 0.0019, 0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026,0.0027, 0.0028, 0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035,0.0036, 0.0037, 0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044,0.0045, 0.0046, 0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053,0.0054, 0.0055, 0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062,0.0063, 0.0064, 0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071,0.0072, 0.0073, 0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080,0.0081, 0.0082, 0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089,0.0090, 0.0091, 0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098,0.0099, 0.0100, 0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107,0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116,0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125,0.0126, 0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134,0.0135, 0.0136, 0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143,0.0144, 0.0145, 0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT.

In some embodiments, the technology provides a method of producing arealtime PCR reagent comprising mixing 0.0001 to 0.1% (w/w) MIT (e.g.,0.001 to 0.01% (w/w) MIT) with at least one of a polymerase, a firstprimer, a second primer, and/or nucleotides. In some embodiments, themethod of producing a realtime PCR reagent comprises mixing 0.0001,0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010,0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019,0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028,0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037,0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046,0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055,0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064,0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073,0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082,0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091,0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100,0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109,0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118,0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127,0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136,0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145,0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT with with at leastone of a polymerase, a first primer, a second primer, and/ornucleotides. In some embodiments, the realtime PCR reagent has a volumegreater than 10 mL (e.g., greater than 100 mL, greater than 1 liter). Insome embodiments, the method of producing a realtime PCR reagentcomprises storing said realtime PCR reagent for at least 1 day, 1 week,or 1 month. In some embodiments, the method of producing a realtime PCRreagent comprises mixing into the realtime PCR reagent a reference dye,a detectably labeled probe, an intercalating dye, a third primer, and/ora fourth primer.

In some embodiments, the technology provides systems for detecting anucleic acid. In some embodiments, the system comprises anoligonucleotide reagent comprising a first primer, a second primer, adetectably labeled probe, nucleotides, and 0.0001 to 0.1% (w/w) MIT; andan activation reagent comprising manganese chloride and 0.0001 to 0.1%(w/w) MIT. In some embodiments, the system further comprises a realtimethermocycler. Some embodiments of the technology provided herein furthercomprise functionalities for collecting, storing, and/or analyzing data.For example, in some embodiments the device comprises a processor, amemory, and/or a database for, e.g., storing and executing instructions,analyzing data, performing calculations using the data, transforming thedata, and storing the data. Moreover, in some embodiments a processor isconfigured to control the device. In some embodiments, the processor isused to initiate and/or terminate the measurement and data collection.In some embodiments, the device comprises a user interface (e.g., akeyboard, buttons, dials, switches, and the like) for receiving userinput that is used by the processor to direct a measurement. In someembodiments, the device further comprises a data output for transmittingdata to an external destination, e.g., a computer, a display, a network,and/or an external storage medium. Some embodiments provide that thedevice is a small, handheld, portable device incorporating thesefeatures and components. In some embodiments, the system furthercomprises a computer configured to determine a Ct value. In someembodiments, the systems further comprise a thermostable polymerase in abuffered solution. In some embodiments, the thermostable polymerase isrTth polymerase.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presenttechnology will become better understood with regard to the followingdrawings. In the figures, “Proclin” refers to PROCLIN 950 (9.5% (w/w)MIT dissolved in water).

FIG. 1 is a plot of RT-PCR data indicating that including MIT in mastermixes reduces the accumulation of non-specific product. The plot showsdRn as a function of cycle number for samples comprising 0, 0.015, 0.05,and 0.1% (w/w) PROCLIN 950 (corresponding to 0, 0.0015, 0.005, and 0.01%(w/w) MIT concentrations, respectively).

FIG. 2 is a plot of RT-PCR data showing that including MIT in RT-PCRmaster mixes reduces the accumulation of non-specific product duringprolonged storage. The plot shows dRn as a function of cycle number forsamples comprising 0, 0.015, 0.05, and 0.1% (w/w) PROCLIN 950(corresponding to 0, 0.0015, 0.005, and 0.01% (w/w) MIT concentrations,respectively).

FIG. 3 is a plot of RT-PCR data indicating that adding MIT to HIV-1assay master mixes enhances RT-PCR performance. The plot shows dRn as afunction of cycle number for samples comprising 50 copies of HIV-1target RNA and either comprising 0.015% (w/w) PROCLIN 950 (0.0015% (w/w)MIT) or comprising no PROCLIN 950.

FIG. 4 is a plot of RT-PCR data indicating that adding MIT to CT/NG(Chlamydia trachomatis and Neisseria gonorrhoeae) master mixes enhancesRT-PCR performance. The plot shows dRn as a function of cycle number forsamples comprising 0.1 inclusion forming units of Chlamydia trachomatisand either comprising 0.015% (w/w) PROCLIN 950 (0.0015% (w/w) MIT) orcomprising no PROCLIN 950.

FIG. 5 is a plot of RT-PCR data indicating that MIT improves stabilityof PCR mastermixes. The plot shows dRn as function of cycle number formaster mixes comprising 0, 0.015, 0.05, and 0.1% (w/w) PROCLIN 950(corresponding to 0, 0.0015, 0.005, and 0.01% (w/w) MIT concentrations,respectively) that were incubated at 2 to 8° C. for 16 hours prior touse in reaction mixtures comprising 50 copies of HIV-1 RNA.

It is to be understood that the figures are not necessarily drawn toscale, nor are the objects in the figures necessarily drawn to scale inrelationship to one another. The figures are depictions that areintended to bring clarity and understanding to various embodiments ofapparatuses, systems, and methods disclosed herein. Wherever possible,the same reference numbers will be used throughout the drawings to referto the same or like parts. Moreover, it should be appreciated that thedrawings are not intended to limit the scope of the present teachings inany way.

DETAILED DESCRIPTION

Provided herein is technology relating to amplification of nucleic acidsand particularly, but not exclusively, to compositions and methods forconducting the polymerase chain reaction and providing reagents forpolymerase chain reactions with improved stability.

In this detailed description of the various embodiments, for purposes ofexplanation, numerous specific details are set forth to provide athorough understanding of the embodiments disclosed. One skilled in theart will appreciate, however, that these various embodiments may bepracticed with or without these specific details. In other instances,structures and devices are shown in block diagram form. Furthermore, oneskilled in the art can readily appreciate that the specific sequences inwhich methods are presented and performed are illustrative and it iscontemplated that the sequences can be varied and still remain withinthe spirit and scope of the various embodiments disclosed herein.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages are expressly incorporated byreference in their entirety for any purpose. Unless defined otherwise,all technical and scientific terms used herein have the same meaning asis commonly understood by one of ordinary skill in the art to which thevarious embodiments described herein belongs. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control. The section headings used herein arefor organizational purposes only and are not to be construed as limitingthe described subject matter in any way.

DEFINITIONS

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

As used herein, the terms “about”, “approximately”, “substantially”, and“significantly” are understood by persons of ordinary skill in the artand will vary to some extent on the context in which they are used. Ifthere are uses of these terms that are not clear to persons of ordinaryskill in the art given the context in which they are used, “about” and“approximately” mean plus or minus less than or equal to 10% (w/w) ofthe particular term and “substantially” and “significantly” mean plus orminus greater than 10% (w/w) of the particular term.

As used herein, disclosure of ranges includes disclosure of all valuesand further divided ranges within the entire range, including endpointsand sub-ranges given for the ranges.

As used herein, the suffix “-free” refers to an embodiment of thetechnology that omits the feature of the base root of the word to which“-free” is appended. That is, the term “X-free” as used herein means“without X”, where X is a feature of the technology omitted in the“X-free” technology. For example, a “calcium-free” composition does notcomprise calcium, a “mixing-free” method does not comprise a mixingstep, etc.

Although the terms “first”, “second”, “third”, etc. may be used hereinto describe various steps, elements, compositions, components, regions,layers, and/or sections, these steps, elements, compositions,components, regions, layers, and/or sections should not be limited bythese terms, unless otherwise indicated. These terms are used todistinguish one step, element, composition, component, region, layer,and/or section from another step, element, composition, component,region, layer, and/or section. Terms such as “first”, “second”, andother numerical terms when used herein do not imply a sequence or orderunless clearly indicated by the context. Thus, a first step, element,composition, component, region, layer, or section discussed herein couldbe termed a second step, element, composition, component, region, layer,or section without departing from technology.

As used herein, “dRn” refers to the magnitude of the fluorescence signalgenerated during a RT-PCR at each time point and/or cycle number. ThedRn value is determined by the following formula (Rn+)−(Rn−). Rn(normalized reporter signal) is the fluorescence emission intensity ofthe reporter dye divided by the fluorescence emission intensity of thepassive reference dye. Rn+ is the Rn value of a reaction containing allcomponents, including the template. Rn− is the Rn value of an unreactedsample. The Rn− value can be obtained from the early cycles of a RT-PCR(e.g., the cycles prior to a significant increase in fluorescence) or ina reaction that does not comprise template.

As used herein, the term “Ct” or “threshold cycle” refers to the cyclenumber at which the fluorescence generated within a reaction crosses athreshold. The Ct is inversely correlated to the logarithm of theinitial copy number. The Ct value assigned to a particular reaction thusreflects the point during the reaction at which a sufficient number ofamplicons have accumulated.

As used herein, the term “intercalating dye” or “dsDNA-binding agent”refers to a molecule that has a higher fluorescence emission when boundto double-stranded DNA (dsDNA) relative to when not bound to dsDNA.Thus, in some real-time PCR, the fluorescence intensity increasesproportionally to dsDNA (amplicon) concentration. An exemplarydsDNA-binding agent is SYBR Green I, which is a fluorogenic minor groovebinding dye that emits little fluorescence when in solution but emits astrong fluorescent signal upon binding to double-stranded DNA. SimilardsDNA-binding fluorescent dyes include EvaGreen and LCGreen.

As used herein, the term “fast PCR” refers to a modified PCR protocolthat is complete in a run time of less than 90 minutes (e.g., 30, 35,40, 45, 50, 55, or 60 minutes or less) due to improvements in amplicondesign, reagent chemistry, thermocycling conditions, and instrumentation(e.g., PCR machines with fast ramping rates).

As used herein, the term “no amplification controls” or “NAC” is acontrol that does not comprise a polymerase enzyme. In mRNA analysis,NAC is a mock reverse transcription containing all the RT-PCR reagentsexcept the reverse transcriptase. If cDNA or genomic DNA is used as atemplate, a reaction mixture lacking Taq polymerase can be included inthe assay as NAC. No product should be synthesized in the NAC.

As used herein, the term “no template controls” or “NTC” is a controlthat does not comprise a sample or target nucleic acid. A NTC includesall the RT-PCR reagents except the RNA template. No product should besynthesized in the NTC.

As used herein, the term “nucleic acid target” or “target template” or“target nucleic acid” refers to the DNA or RNA sequence that is or isintended to be amplified. The nucleic acid sample used to amplify thetarget sequence is called template. In a typical qPCR, the cDNA sampleincluded in the reaction is the template.

As used herein, the term “realtime PCR” refers to the continuouscollection of fluorescent signal from polymerase chain reactionthroughout cycles.

As used herein, the term “threshold” refers to the fluorescent signallevel that is considered to be above background level or noise. In someembodiments, the threshold is 10× the standard deviation of Rn for theearly PCR cycles and/or is set in the region associated with anexponential growth of PCR product and not as high as the linear orplateau sections of the curve. In some embodiments, the threshold isabove the highest baseline signal level.

As used herein, a “nucleic acid” shall mean any nucleic acid molecule,including, without limitation, DNA, RNA, and hybrids thereof. Thenucleic acid bases that form nucleic acid molecules can be the bases A,C, G, T and U, as well as derivatives thereof. Derivatives of thesebases are well known in the art. The term should be understood toinclude, as equivalents, analogs of either DNA or RNA made fromnucleotide analogs. The term as used herein also encompasses cDNA, thatis complementary, or copy, DNA produced from an RNA template, forexample, by the action of a reverse transcriptase.

Reference to a base, a nucleotide, or to another molecule may be in thesingular or plural. That is, “a base” may refer to a single molecule ofthat base or to a plurality of the base, e.g., in a solution.

A “polynucleotide”, “nucleic acid”, or “oligonucleotide” refers to alinear polymer of nucleosides (including deoxyribonucleosides,ribonucleosides, or analogs thereof) joined by internucleosidiclinkages. Typically, a polynucleotide comprises at least threenucleosides. Usually, oligonucleotides range in size from a fewmonomeric units, e.g. 3-4, to several hundreds of monomeric units.Whenever a polynucleotide such as an oligonucleotide is represented by asequence of letters, such as “ATGCCTG”, it will be understood that thenucleotides are in 5′ to 3′ order from left to right and that “A” or “a”denotes deoxyadenosine, “C” or “c” denotes deoxycytidine, “G” or “g”denotes deoxyguanosine, and “T” or “t” denotes thymidine, unlessotherwise noted. The letters A, C, G, and T may be used to refer to thebases themselves, to nucleosides, or to nucleotides comprising thebases, as is standard in the art.

In some embodiments, nucleic acids comprise a universal or modified basesuch as deoxyinosine, inosine, 7-deaza-2′-deoxyinosine,2-aza-2′-deoxyinosine, 2′-0-Me inosine, 2′-F inosine, deoxy3-nitropyrrole, 3-nitropyrrole, 2′-O-Me 3-nitropyrrole, 2′-F3-nitropyrrole, 1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, deoxy5-nitroindole, 5-nitroindole, 2′-O-Me 5-nitroindole, 2′-F 5-nitroindole,deoxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy4-aminobenzimidazole, 4-aminobenzimidazole, deoxy nebularine, 2′-Fnebularine, 2′-F 4-nitrobenzimidazole, PNA-5-introindole,PNA-nebularine, PNA-inosine, PNA-4-nitrobenzimidazole,PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebularine,morpholino-inosine, morpholino-4-nitrobenzimidazole,morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole,phosphoramidate-nebularine, phosphoramidate-inosine,phosphoramidate-4-nitrobenzimidazole, phosphoramidate-3-nitropyrrole,2′-O-methoxyethyl inosine, 2′-O-methoxyethyl nebularine,2′-O-methoxyethyl 5-nitroindole, 2′-O-methoxyethyl4-nitro-benzimidazole, 2′-O-methoxyethyl 3-nitropyrrole, andcombinations thereof.

As used herein, “complementary” generally refers to specific nucleotideduplexing to form canonical Watson-Crick base pairs, as is understood bythose skilled in the art. However, complementary also includesbase-pairing of nucleotide analogs that are capable of universalbase-pairing with A, T, G or C nucleotides and locked nucleic acids thatenhance the thermal stability of duplexes. One skilled in the art willrecognize that hybridization stringency is a determinant in the degreeof match or mismatch in the duplex formed by hybridization.

As used herein, the term “polymerase” refers to an enzyme generally forjoining 3′-OH 5′-triphosphate nucleotides, oligomers, and their analogs.Polymerases include, but are not limited to, DNA-dependent DNApolymerases, DNA-dependent RNA polymerases, RNA-dependent DNApolymerases, RNA-dependent RNA polymerases, T7 DNA polymerase, T3 DNApolymerase, T4 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6RNA polymerase, DNA polymerase 1, Klenow fragment, Thermophilusaquaticus (Taq) DNA polymerase, Thermus thermophiles (Tth) DNApolymerase (e.g., recombinant Tth (rTth) polymerase; see, e.g., Abual-Soud and Radstrom (1998) “Capacity of nine thermostable DNApolymerases to mediate DNA amplification in the presence ofPCR-Inhibiting samples” Appl Environ Microbiol. 64: 3748-53; Myers andGelfand (1991) “Reverse transcription and DNA amplification by athermus-thermophilus DNA-polymerase” Biochemistry 30: 7661-66, each ofwhich is incorporated herein by reference), Vent DNA polymerase (NewEngland Biolabs), Deep Vent DNA polymerase (New England Biolabs),Bacillus stearothermophilus (Bst) DNA polymerase, DNA Polymerase LargeFragment, Stoeffel Fragment, 9° N DNA Polymerase, 9° Nm polymerase,Pyrococcus furiosis (Pfu) DNA Polymerase, Thermus filifbrmis (Tfl) DNAPolymerase, RepliPHI Phi29 Polymerase, Thermococcus litoralis (Tli) DNApolymerase, eukaryotic DNA polymerase beta, telomerase, Therminatorpolymerase (New England Biolabs), KOD HiFi. DNA polymerase (Novagen),KOD1 DNA polymerase, Q-beta replicase, terminal transferase, AMV reversetranscriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase,HIV- 1 reverse transcriptase, novel polymerases discovered bybioprospecting and/or molecular evolution, and polymerases cited in U.S.Pat. Appl. Pub. No. 2007/0048748 and in U.S. Pat. Nos. 6,329,178;6,602,695; and 6,395,524. These polymerases include wild-type, mutantisoforms, and genetically engineered variants such as exo-polymerases;polymerases with minimized, undetectable, and/or decreased 3′→5′proofreading exonuclease activity, and other mutants, e.g., thattolerate labeled nucleotides and incorporate them into a strand ofnucleic acid. In some embodiments, the polymerase is designed for use,e.g., in real-time PCR, high fidelity PCR, next-generation DNAsequencing, fast PCR, hot start PCR, crude sample PCR, robust PCR,and/or molecular diagnostics. Such enzymes are available from manycommercial suppliers, e.g., Kapa Enzymes, Finnzymes, Promega,Invitrogen, Life Technologies, Thermo Scientific, Qiagen, Roche, etc. Insome embodiments, the polymerase has 5′→3′ exonuclease activity and canthus degrade a nucleic acid from a 5′ end in addition to catalyzingsynthesis of a nucleic acid from a 3′-OH of a nucleic acid (e.g., from aprimer, e.g., a hairpin primer). In some embodiments the polymerase(e.g., a high-fidelity polymerase) comprises a proof-reading activity, a3′ exonuclease activity, and/or a strand displacement activity, butlacks a 5′ exonuclease activity.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product that is complementary to a nucleic acid strand isinduced, (e.g., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method. Asused herein, the single stranded (e.g., amplicon-specific) portion of ahairpin primer may serve to prime the synthesis of a nucleic acid.

As used herein, the term “annealing” or “priming” refers to theapposition of an oligodeoxynucleotide or nucleic acid to a templatenucleic acid, whereby the apposition enables the polymerase topolymerize nucleotides into a nucleic acid molecule that iscomplementary to the template nucleic acid or a portion thereof. Theterm “hybridizing” as used herein refers to the formation of adouble-stranded nucleic acid from complementary single stranded nucleicacids. There is no intended distinction between the terms “annealing”and “hybridizing”, and these terms will be used interchangeably. Thesequences of primers may comprise some mismatches, so long as they canbe hybridized with templates and serve as primers. The term“substantially complementary” is used herein to signify that the primeris sufficiently complementary to hybridize selectively to a templatenucleic acid sequence under the designated annealing conditions orstringent conditions, such that the annealed primer can be extended by apolymerase to form a complementary copy of the template.

As used herein, the term “sample” is used in its broadest sense. In onesense it can refer to an animal cell or tissue. In another sense, it ismeant to include a specimen or culture obtained from any source, as wellas biological and environmental samples. Biological samples may beobtained from plants or animals (including humans) and encompass fluids,solids, tissues, and gases. Environmental samples include environmentalmaterial such as surface matter, soil, water, and industrial samples.These examples are not to be construed as limiting the sample typesapplicable to the present invention. In particular embodiments, a samplecomprises a target nucleic acid.

Description

The compound 2-methyl-4-isothiazolin-3-one (MIT) is a biocidal compoundknown to suppress microbial respiration through inhibition of specificKrebs cycle enzymes. MIT has a CAS Number 2682-20-4, Empirical Formulaof C4H5NOS, and molecular weight 115.15. MIT has a structure accordingto

MIT is the active ingredient in the antimicrobial agent PROCLIN 950(9.5% (w/w) MIT dissolved in water), which is used to prevent microbialgrowth. Embodiments of the technology provided herein relate to use ofMIT to improve PCR. In some embodiments, the technology comprises use ofMIT at a concentration that does not have biocidal activity, or has pooror undesirable levels of biocidal activity, in a reagent used foramplification of a nucleic acid (e.g., PCR).

In some embodiments, the technology provides a composition comprisingone or more components of a PCR (e.g., polymerase, primers, probes,nucleotides (e.g., one or more of dATP, dCTP, dGTP, and dTTP monomers),intercalating dye, water, buffer, sample, and/or target nucleic acid)and MIT. In some embodiments, the technology provides a compositioncomprising a polymerase and MIT. In some embodiments, the technologyprovides a composition comprising a probe and MIT. In some embodiments,the technology provides a composition comprising at intercalating dyeand MIT. In some embodiments, the technology provides a compositioncomprising nucleotides (e.g., one or more of dATP, dCTP, dGTP, and dTTPmonomers) and MIT. In some embodiments, the technology provides acomposition comprising a polymerase, nucleotides (e.g., one or more ofdATP, dCTP, dGTP, and dTTP monomers), and MIT. In some embodiments, thetechnology provides a composition comprising a polymerase, nucleotides(e.g., one or more of dATP, dCTP, dGTP, and dTTP monomers), a probe, andMIT. In some embodiments, the technology provides a compositioncomprising a polymerase, nucleotides (e.g., one or more of dATP, dCTP,dGTP, and dTTP monomers), an intercalating dye, and MIT.

During the development of embodiments of the technology provided herein,experiments were conducted to test concentrations of MIT in RT-PCR. Datacollected during the experiments indicated that nucleic acidamplification (e.g., PCR (e.g., RT-PCR)) was improved by adding MIT tothe reaction mixture. In particular, reaction mixtures were producedcomprising 0.0015, 0.005, and 0.01% (w/w) MIT. Accordingly, in someembodiments, the technology provides a composition comprisingapproximately 0.001 to 0.002% (w/w) MIT. In some embodiments, thetechnology provides a composition comprising approximately 0.0001 to0.01% (w/w) MIT. In some embodiments, the technology provides acomposition comprising approximately 0.0045 to 0.0055% (w/w) MIT. Insome embodiments, the technology provides a composition comprisingapproximately 0.001 to 0.1% (w/w) MIT. In some embodiments, thetechnology provides a composition for nucleic acid amplificationcomprising 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007,0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016,0.0017, 0.0018, 0.0019, 0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025,0.0026, 0.0027, 0.0028, 0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034,0.0035, 0.0036, 0.0037, 0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043,0.0044, 0.0045, 0.0046, 0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052,0.0053, 0.0054, 0.0055, 0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061,0.0062, 0.0063, 0.0064, 0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070,0.0071, 0.0072, 0.0073, 0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079,0.0080, 0.0081, 0.0082, 0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088,0.0089, 0.0090, 0.0091, 0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097,0.0098, 0.0099, 0.0100, 0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106,0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115,0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124,0.0125, 0.0126, 0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133,0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142,0.0143, 0.0144, 0.0145, 0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w)MIT.

In some embodiments, MIT is provided as a mixture in water (e.g., as asolution comprising 9.5% (w/w) MIT dissolved in water and, optionally,other components (e.g., PROCLIN 950)). In some embodiments, thetechnology provides a composition for nucleic acid amplificationcomprising 0.010, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017,0.018, 0.019, 0.020, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027,0.028, 0.029, 0.030, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037,0.038, 0.039, 0.040, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047,0.048, 0.049, or 0.050 PROCLIN 950.

In some embodiments, the technology provides a composition having avolume ranging from nanoliters to microliters (e.g., a PCR or RT-PCRreaction) to milliliters to centiliters (e.g., a PCR or RT-PCR mastermix) to deciliters to liters to decaliters or more (e.g., PCR or RT-PCRreagents produced at a commercial scale). Accordingly, in someembodiments, the technology provides a composition having a volume of 1,5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or1000 nl and comprising one or more components of a PCR (e.g.,polymerase, primers, probes, nucleotides (e.g., one or more of dATP,dCTP, dGTP, and dTTP monomers), intercalating dye, water, buffer,sample, and/or target nucleic acid) and 0.0001, 0.0002, 0.0003, 0.0004,0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013,0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0020, 0.0021, 0.0022,0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028, 0.0029, 0.0030, 0.0031,0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037, 0.0038, 0.0039, 0.0040,0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046, 0.0047, 0.0048, 0.0049,0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055, 0.0056, 0.0057, 0.0058,0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064, 0.0065, 0.0066, 0.0067,0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073, 0.0074, 0.0075, 0.0076,0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082, 0.0083, 0.0084, 0.0085,0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091, 0.0092, 0.0093, 0.0094,0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100, 0.0101, 0.0102, 0.0103,0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112,0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121,0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129, 0.0130,0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139,0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145, 0.0146, 0.0147, 0.0148,0.0149, or 0.0150% (w/w) MIT.

In some embodiments, the technology provides a composition having avolume of 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, or 1000 μl and comprising one or more components of a PCR(e.g., polymerase, primers, probes, nucleotides (e.g., one or more ofdATP, dCTP, dGTP, and dTTP monomers), intercalating dye, water, buffer,sample, and/or target nucleic acid) and 0.0001, 0.0002, 0.0003, 0.0004,0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013,0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0020, 0.0021, 0.0022,0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028, 0.0029, 0.0030, 0.0031,0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037, 0.0038, 0.0039, 0.0040,0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046, 0.0047, 0.0048, 0.0049,0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055, 0.0056, 0.0057, 0.0058,0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064, 0.0065, 0.0066, 0.0067,0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073, 0.0074, 0.0075, 0.0076,0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082, 0.0083, 0.0084, 0.0085,0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091, 0.0092, 0.0093, 0.0094,0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100, 0.0101, 0.0102, 0.0103,0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112,0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121,0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129, 0.0130,0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139,0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145, 0.0146, 0.0147, 0.0148,0.0149, or 0.0150% (w/w) MIT.

In some embodiments, the technology provides a composition having avolume of 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, or 1000 ml and comprising one or more components of a PCR(e.g., polymerase, primers, probes, nucleotides (e.g., one or more ofdATP, dCTP, dGTP, and dTTP monomers), intercalating dye, water, buffer,sample, and/or target nucleic acid) and 0.0001, 0.0002, 0.0003, 0.0004,0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013,0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0020, 0.0021, 0.0022,0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028, 0.0029, 0.0030, 0.0031,0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037, 0.0038, 0.0039, 0.0040,0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046, 0.0047, 0.0048, 0.0049,0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055, 0.0056, 0.0057, 0.0058,0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064, 0.0065, 0.0066, 0.0067,0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073, 0.0074, 0.0075, 0.0076,0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082, 0.0083, 0.0084, 0.0085,0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091, 0.0092, 0.0093, 0.0094,0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100, 0.0101, 0.0102, 0.0103,0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112,0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121,0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129, 0.0130,0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139,0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145, 0.0146, 0.0147, 0.0148,0.0149, or 0.0150% (w/w) MIT.

In some embodiments, the technology provides a composition having avolume of 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, or 1000 L and comprising one or more components of a PCR(e.g., polymerase, primers, probes, nucleotides (e.g., one or more ofdATP, dCTP, dGTP, and dTTP monomers), intercalating dye, water, buffer,sample, and/or target nucleic acid) and 0.0001, 0.0002, 0.0003, 0.0004,0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013,0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0020, 0.0021, 0.0022,0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028, 0.0029, 0.0030, 0.0031,0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037, 0.0038, 0.0039, 0.0040,0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046, 0.0047, 0.0048, 0.0049,0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055, 0.0056, 0.0057, 0.0058,0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064, 0.0065, 0.0066, 0.0067,0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073, 0.0074, 0.0075, 0.0076,0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082, 0.0083, 0.0084, 0.0085,0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091, 0.0092, 0.0093, 0.0094,0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100, 0.0101, 0.0102, 0.0103,0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112,0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121,0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129, 0.0130,0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139,0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145, 0.0146, 0.0147, 0.0148,0.0149, or 0.0150% (w/w) MIT.

In some embodiments, adding MIT to a PCR reaction mixture increases thesignal of a realtime PCR (e.g., realtime RT-PCR) by 1.1× to 10× or more(e.g., 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2.0×, 2.1×,2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, 3.0×, 3.1×, 3.2×, 3.3×,3.4×, 3.5×, 3.6×, 3.7×, 3.8×, 3.9×, 4.0×, 4.1×, 4.2×, 4.3×, 4.4×, 4.5×,4.6×, 4.7×, 4.8×, 4.9×, 5.0×, 5.1×, 5.2×, 5.3×, 5.4×, 5.5×, 5.6×, 5.7×,5.8×, 5.9×, 6.0×, 6.1×, 6.2×, 6.3×, 6.4×, 6.5×, 6.6×, 6.7×, 6.8×, 6.9×,7.0×, 7.1×, 7.2×, 7.3×, 7.4×, 7.5×, 7.6×, 7.7×, 7.8×, 7.9×, 8.0×, 8.1×,8.2×, 8.3×, 8.4×, 8.5×, 8.6×, 8.7×, 8.8×, 8.9×, 9.0×, 9.1×, 9.2×, 9.3×,9.4×, 9.5×, 9.6×, 9.7×, 9.8×, 9.9×, or 10.0× or more) relative to a PCRreaction that does not comprise MIT.

In some embodiments, adding MIT to a PCR reaction mixture decreases theCt indicating the presence of a target nucleic acid the signal of arealtime PCR (e.g., realtime RT-PCR) by 1-20 cycles (e.g., 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7,10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9,12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1,13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3,14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0, 15.1, 15.2, 15.3, 15.4, 15.5,15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7,16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9,18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1,19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, or 20.0 cycles) relativeto a PCR reaction that does not comprise MIT.

In some embodiments, adding MIT to a PCR reaction mixture decreases theamount of non-specific product by 1 to 100% (e.g., 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5,10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5,16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5,22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, 26.0, 26.5, 27.0, 27.5,28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5,34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5, 38.0, 38.5, 39.0, 39.5,40.0, 40.5, 41.0, 41.5, 42.0, 42.5, 43.0, 43.5, 44.0, 44.5, 45.0, 45.5,46.0, 46.5, 47.0, 47.5, 48.0, 48.5, 49.0, 49.5, 50.0, 50.5, 51.0, 51.5,52.0, 52.5, 53.0, 53.5, 54.0, 54.5, 55.0, 55.5, 56.0, 56.5, 57.0, 57.5,58.0, 58.5, 59.0, 59.5, 60.0, 60.5, 61.0, 61.5, 62.0, 62.5, 63.0, 63.5,64.0, 64.5, 65.0, 65.5, 66.0, 66.5, 67.0, 67.5, 68.0, 68.5, 69.0, 69.5,70.0, 70.5, 71.0, 71.5, 72.0, 72.5, 73.0, 73.5, 74.0, 74.5, 75.0, 75.5,76.0, 76.5, 77.0, 77.5, 78.0, 78.5, 79.0, 79.5, 80.0, 80.5, 81.0, 81.5,82.0, 82.5, 83.0, 83.5, 84.0, 84.5, 85.0, 85.5, 86.0, 86.5, 87.0, 87.5,88.0, 88.5, 89.0, 89.5, 90.0, 90.5, 91.0, 91.5, 92.0, 92.5, 93.0, 93.5,94.0, 94.5, 95.0, 95.5, 96.0, 96.5, 97.0, 97.5, 98.0, 98.5, 99.0, 99.5,100.0%).

In some embodiments, adding MIT to a PCR reaction mixture delays theformation of non-specific products that fluoresce above a threshold by1-20 cycles (e.g., 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2,10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4,11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6,12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8,13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0,15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2,16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4,17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6,18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8,19.9, or 20.0 cycles) relative to a PCR reaction that does not compriseMIT.

In some embodiments, the technology provides methods for producing acomposition for amplifying nucleic acids (e.g., by PCR (e.g., byRT-PCR)). For example, in some embodiments, methods comprise providing,producing, and/or obtaining a composition comprising one or morecomponents of a PCR (e.g., polymerase, primers, probes, nucleotides(e.g., one or more of dATP, dCTP, dGTP, and dTTP monomers),intercalating dye, water, buffer, sample, and/or target nucleic acid)and MIT. In some embodiments, MIT is provided by providing, producing,and/or obtaining a composition comprising PROCLIN 950.

In some embodiments, methods comprise mixing a composition comprisingone or more components of a PCR (e.g., polymerase, primers, probes,nucleotides (e.g., one or more of dATP, dCTP, dGTP, and dTTP monomers),intercalating dye, water, buffer, sample, and/or target nucleic acid)and 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008,0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017,0.0018, 0.0019, 0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026,0.0027, 0.0028, 0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035,0.0036, 0.0037, 0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044,0.0045, 0.0046, 0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053,0.0054, 0.0055, 0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062,0.0063, 0.0064, 0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071,0.0072, 0.0073, 0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080,0.0081, 0.0082, 0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089,0.0090, 0.0091, 0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098,0.0099, 0.0100, 0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107,0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116,0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125,0.0126, 0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134,0.0135, 0.0136, 0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143,0.0144, 0.0145, 0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT.

In some embodiments, methods comprise adding a sample (e.g., a nucleicacid) to a composition comprising MIT. In some embodiments, methodscomprise adding a primer to a composition comprising MIT. In someembodiments, methods comprise adding a probe to a composition comprisingMIT. In some embodiments, methods comprise adding an intercalating dyeto a composition comprising MIT. In some embodiments, methods compriseadding a polymerase to a composition comprising MIT. In someembodiments, methods comprise adding one or more components of a PCR(e.g., polymerase, primers, probes, nucleotides (e.g., one or more ofdATP, dCTP, dGTP, and dTTP monomers), intercalating dye, water, buffer,sample, and/or target nucleic acid) to a composition comprising 0.0001,0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010,0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019,0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028,0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037,0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046,0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055,0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064,0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073,0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082,0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091,0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100,0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109,0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118,0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127,0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136,0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145,0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT.

In some embodiments, methods comprise adding a composition comprisingMIT to a composition comprising a sample (e.g., a nucleic acid). In someembodiments, methods comprise adding a composition comprising MIT to acomposition comprising a primer. In some embodiments, methods compriseadding a composition comprising MIT to a composition comprising a probe.In some embodiments, methods comprise adding a composition comprisingMIT to a composition comprising an intercalating dye. In someembodiments, methods comprise adding a composition comprising MIT to acomposition comprising a polymerase. In some embodiments, methodscomprise adding a composition comprising 0.0001, 0.0002, 0.0003, 0.0004,0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013,0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0020, 0.0021, 0.0022,0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028, 0.0029, 0.0030, 0.0031,0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037, 0.0038, 0.0039, 0.0040,0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046, 0.0047, 0.0048, 0.0049,0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055, 0.0056, 0.0057, 0.0058,0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064, 0.0065, 0.0066, 0.0067,0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073, 0.0074, 0.0075, 0.0076,0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082, 0.0083, 0.0084, 0.0085,0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091, 0.0092, 0.0093, 0.0094,0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100, 0.0101, 0.0102, 0.0103,0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112,0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121,0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129, 0.0130,0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139,0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145, 0.0146, 0.0147, 0.0148,0.0149, or 0.0150% (w/w) MIT to a composition comprising one or morecomponents of a PCR (e.g., polymerase, primers, probes, nucleotides(e.g., one or more of dATP, dCTP, dGTP, and dTTP monomers),intercalating dye, water, buffer, sample, and/or target nucleic acid).

In some embodiments, methods comprise storing a composition comprisingone or more components of a PCR (e.g., polymerase, primers, probes,nucleotides (e.g., one or more of dATP, dCTP, dGTP, and dTTP monomers),intercalating dye, water, buffer, sample, and/or target nucleic acid)and 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008,0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017,0.0018, 0.0019, 0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026,0.0027, 0.0028, 0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035,0.0036, 0.0037, 0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044,0.0045, 0.0046, 0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053,0.0054, 0.0055, 0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062,0.0063, 0.0064, 0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071,0.0072, 0.0073, 0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080,0.0081, 0.0082, 0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089,0.0090, 0.0091, 0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098,0.0099, 0.0100, 0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107,0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116,0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125,0.0126, 0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134,0.0135, 0.0136, 0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143,0.0144, 0.0145, 0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT for1 to 60 minutes (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0,12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0,18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0,24.5, 25.0, 25.5, 26.0, 26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0,30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0,36.5, 37.0, 37.5, 38.0, 38.5, 39.0, 39.5, 40.0, 40.5, 41.0, 41.5, 42.0,42.5, 43.0, 43.5, 44.0, 44.5, 45.0, 45.5, 46.0, 46.5, 47.0, 47.5, 48.0,48.5, 49.0, 49.5, 50.0, 50.5, 51.0, 51.5, 52.0, 52.5, 53.0, 53.5, 54.0,54.5, 55.0, 55.5, 56.0, 56.5, 57.0, 57.5, 58.0, 58.5, 59.0, 59.5, or60.0 minutes), 1 to 24 hours (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0,17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0,23.5, or 24.0 hours), 1 to 30 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 days), 1 to 12 months (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5,11.0, 11.5, or 12.0 months), or 1 to 10 years (e.g., 1.00, 1.25, 1.50,1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, 4.50,4.75, 5.00, 5.25, 5.50, 5.75, 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50,7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, or 10.00 years).

In some embodiments, methods comprise storing a composition comprisingone or more components of a PCR (e.g., polymerase, primers, probes,nucleotides (e.g., one or more of dATP, dCTP, dGTP, and dTTP monomers),intercalating dye, water, buffer, sample, and/or target nucleic acid)and 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008,0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017,0.0018, 0.0019, 0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026,0.0027, 0.0028, 0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035,0.0036, 0.0037, 0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044,0.0045, 0.0046, 0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053,0.0054, 0.0055, 0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062,0.0063, 0.0064, 0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071,0.0072, 0.0073, 0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080,0.0081, 0.0082, 0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089,0.0090, 0.0091, 0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098,0.0099, 0.0100, 0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107,0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116,0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125,0.0126, 0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134,0.0135, 0.0136, 0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143,0.0144, 0.0145, 0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT at−5 to 30° C. (e.g., −5.0, −4.5, −4.0, −3.5, −3.0, −2.5, −2.0, −1.5,−1.0, −0.5, 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0,12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0,18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0,24.5, 25.0, 25.5, 26.0, 26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, or30.0° C.).

In some embodiments, methods comprise storing a composition comprisingone or more components of a PCR (e.g., polymerase, primers, probes,nucleotides (e.g., one or more of dATP, dCTP, dGTP, and dTTP monomers),intercalating dye, water, buffer, sample, and/or target nucleic acid)and 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008,0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017,0.0018, 0.0019, 0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026,0.0027, 0.0028, 0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035,0.0036, 0.0037, 0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044,0.0045, 0.0046, 0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053,0.0054, 0.0055, 0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062,0.0063, 0.0064, 0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071,0.0072, 0.0073, 0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080,0.0081, 0.0082, 0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089,0.0090, 0.0091, 0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098,0.0099, 0.0100, 0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107,0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116,0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125,0.0126, 0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134,0.0135, 0.0136, 0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143,0.0144, 0.0145, 0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT at−5 to 30° C. (e.g., −5.0, −4.5, −4.0, −3.5, −3.0, −2.5, −2.0, −1.5,−1.0, −0.5, 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0,12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0,18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0,24.5, 25.0, 25.5, 26.0, 26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, or30.0° C.) for 1 to 60 minutes (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0,17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0,23.5, 24.0, 24.5, 25.0, 25.5, 26.0, 26.5, 27.0, 27.5, 28.0, 28.5, 29.0,29.5, 30.0, 30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0,35.5, 36.0, 36.5, 37.0, 37.5, 38.0, 38.5, 39.0, 39.5, 40.0, 40.5, 41.0,41.5, 42.0, 42.5, 43.0, 43.5, 44.0, 44.5, 45.0, 45.5, 46.0, 46.5, 47.0,47.5, 48.0, 48.5, 49.0, 49.5, 50.0, 50.5, 51.0, 51.5, 52.0, 52.5, 53.0,53.5, 54.0, 54.5, 55.0, 55.5, 56.0, 56.5, 57.0, 57.5, 58.0, 58.5, 59.0,59.5, or 60.0 minutes), 1 to 24 hours (e.g., 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0,10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0,16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0,22.5, 23.0, 23.5, or 24.0 hours), 1 to 30 days (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 days), 1 to 12 months (e.g., 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5,10.0, 10.5, 11.0, 11.5, or 12.0 months), or 1 to 10 years (e.g., 1.00,1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00,4.25, 4.50, 4.75, 5.00, 5.25, 5.50, 5.75, 6.00, 6.25, 6.50, 6.75, 7.00,7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, or10.00 years).

In some embodiments, methods comprise storing a composition having avolume of 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, or 1000 nl, μl, ml, or L and comprising one or more componentsof a PCR (e.g., polymerase, primers, probes, nucleotides (e.g., one ormore of dATP, dCTP, dGTP, and dTTP monomers), intercalating dye, water,buffer, sample, and/or target nucleic acid) and 0.0001, 0.0002, 0.0003,0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012,0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0020, 0.0021,0.0022, 0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028, 0.0029, 0.0030,0.0031, 0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037, 0.0038, 0.0039,0.0040, 0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046, 0.0047, 0.0048,0.0049, 0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055, 0.0056, 0.0057,0.0058, 0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064, 0.0065, 0.0066,0.0067, 0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073, 0.0074, 0.0075,0.0076, 0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082, 0.0083, 0.0084,0.0085, 0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091, 0.0092, 0.0093,0.0094, 0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100, 0.0101, 0.0102,0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111,0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120,0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129,0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138,0.0139, 0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145, 0.0146, 0.0147,0.0148, 0.0149, or 0.0150% (w/w) MIT for 1 to 60 minutes (e.g., 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0,14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0,20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, 26.0,26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5, 32.0,32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5, 38.0,38.5, 39.0, 39.5, 40.0, 40.5, 41.0, 41.5, 42.0, 42.5, 43.0, 43.5, 44.0,44.5, 45.0, 45.5, 46.0, 46.5, 47.0, 47.5, 48.0, 48.5, 49.0, 49.5, 50.0,50.5, 51.0, 51.5, 52.0, 52.5, 53.0, 53.5, 54.0, 54.5, 55.0, 55.5, 56.0,56.5, 57.0, 57.5, 58.0, 58.5, 59.0, 59.5, or 60.0 minutes), 1 to 24hours (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0,13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0,19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, or 24.0 hours), 1to 30 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days), 1 to 12months (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, or 12.0months), or 1 to 10 years (e.g., 1.00, 1.25, 1.50, 1.75, 2.00, 2.25,2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, 4.50, 4.75, 5.00, 5.25,5.50, 5.75, 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25,8.50, 8.75, 9.00, 9.25, 9.50, 9.75, or 10.00 years).

In some embodiments, methods comprise storing a composition having avolume of 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, or 1000 nl, μl, ml, or L and comprising one or more componentsof a PCR (e.g., polymerase, primers, probes, nucleotides (e.g., one ormore of dATP, dCTP, dGTP, and dTTP monomers), intercalating dye, water,buffer, sample, and/or target nucleic acid) and 0.0001, 0.0002, 0.0003,0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012,0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0020, 0.0021,0.0022, 0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028, 0.0029, 0.0030,0.0031, 0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037, 0.0038, 0.0039,0.0040, 0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046, 0.0047, 0.0048,0.0049, 0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055, 0.0056, 0.0057,0.0058, 0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064, 0.0065, 0.0066,0.0067, 0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073, 0.0074, 0.0075,0.0076, 0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082, 0.0083, 0.0084,0.0085, 0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091, 0.0092, 0.0093,0.0094, 0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100, 0.0101, 0.0102,0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111,0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120,0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129,0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138,0.0139, 0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145, 0.0146, 0.0147,0.0148, 0.0149, or 0.0150% (w/w) MIT at −5 to 30° C. (e.g., −5.0, −4.5,−4.0, −3.5, −3.0, −2.5, −2.0, −1.5, −1.0, −0.5, 0.0, 0.5, 1.0, 1.5, 2.0,2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0,9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0,15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0,21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, 26.0, 26.5, 27.0,27.5, 28.0, 28.5, 29.0, 29.5, or 30.0° C.) for 1 to 60 minutes (e.g.,1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5,14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5,20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5,26.0, 26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5,32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5,38.0, 38.5, 39.0, 39.5, 40.0, 40.5, 41.0, 41.5, 42.0, 42.5, 43.0, 43.5,44.0, 44.5, 45.0, 45.5, 46.0, 46.5, 47.0, 47.5, 48.0, 48.5, 49.0, 49.5,50.0, 50.5, 51.0, 51.5, 52.0, 52.5, 53.0, 53.5, 54.0, 54.5, 55.0, 55.5,56.0, 56.5, 57.0, 57.5, 58.0, 58.5, 59.0, 59.5, or 60.0 minutes), 1 to24 hours (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5,13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5,19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, or 24.0hours), 1 to 30 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30days), 1 to 12 months (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5,5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,11.5, or 12.0 months), or 1 to 10 years (e.g., 1.00, 1.25, 1.50, 1.75,2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, 4.50, 4.75,5.00, 5.25, 5.50, 5.75, 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75,8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, or 10.00 years).

In some embodiments, the technology provides a method comprisingaliquoting a composition comprising one or more components of a PCR(e.g., polymerase, primers, probes, nucleotides (e.g., one or more ofdATP, dCTP, dGTP, and dTTP monomers), intercalating dye, water, buffer,sample, and/or target nucleic acid) and 0.0001, 0.0002, 0.0003, 0.0004,0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013,0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0020, 0.0021, 0.0022,0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028, 0.0029, 0.0030, 0.0031,0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037, 0.0038, 0.0039, 0.0040,0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046, 0.0047, 0.0048, 0.0049,0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055, 0.0056, 0.0057, 0.0058,0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064, 0.0065, 0.0066, 0.0067,0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073, 0.0074, 0.0075, 0.0076,0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082, 0.0083, 0.0084, 0.0085,0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091, 0.0092, 0.0093, 0.0094,0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100, 0.0101, 0.0102, 0.0103,0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112,0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121,0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129, 0.0130,0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139,0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145, 0.0146, 0.0147, 0.0148,0.0149, or 0.0150% (w/w) MIT into a plurality of smaller volumes.

In some embodiments, methods comprise thermocycling a composition asdescribed herein, e.g., as provided by a method described herein. Insome embodiments, methods comprise recording fluorescence emission as afunction of time and/or cycle number, e.g., to provide realtime PCRand/or realtime RT-PCR data.

In some embodiments, sample preparation and assay performance areautomated (e.g., using automated sample handling, amplification, andanalysis systems). In some embodiments, commercially available systems(e.g., available from Abbott, Abbott Park, Ill.; see, e.g., U.S. Pat.No. 8,703,445; incorporated herein by reference) are utilized.

In some embodiments, the technology comprises software and a computerprocessor and display screen (e.g., computer, laptop, smart phone,tablet, etc.) for analyzing and displaying data.

In some embodiments, assay components are provided in the form of asystem or kit. In some embodiments, kits comprise assay reagents (e.g.,nucleic acid primers or probes, buffers, controls, dNTPs, etc.),controls, software, instructions, etc. In some embodiments, reagents areprovided in one or more separate containers (e.g., vials, wells, tubes,etc.). For example, in some embodiments, reagents are each provided inseparate containers.

In some embodiments, the technology provides a kit comprising one ormore compositions as described herein (e.g., a composition comprisingone or more components of a PCR (e.g., polymerase, primers, probes,nucleotides (e.g., one or more of dATP, dCTP, dGTP, and dTTP monomers),intercalating dye, water, buffer, sample, and/or target nucleic acid)),wherein at least one of the compositions of the kit comprises 0.0001,0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010,0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019,0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028,0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037,0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046,0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055,0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064,0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073,0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082,0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091,0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100,0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109,0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118,0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127,0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136,0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145,0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT. In someembodiments, one or more, two or more, three or more, or four or morecompositions comprise MIT. In some embodiments, one compositioncomprises MIT.

In some embodiments, the technology provides a system comprising one ormore compositions as described herein (e.g., a composition comprisingone or more components of a PCR (e.g., polymerase, primers, probes,nucleotides (e.g., one or more of dATP, dCTP, dGTP, and dTTP monomers),intercalating dye, water, buffer, sample, and/or target nucleic acid)),wherein at least one of the compositions of the kit comprises 0.0001,0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010,0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019,0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028,0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037,0.0038, 0.0039, 0.0040, 0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046,0.0047, 0.0048, 0.0049, 0.0050, 0.0051, 0.0052, 0.0053, 0.0054, 0.0055,0.0056, 0.0057, 0.0058, 0.0059, 0.0060, 0.0061, 0.0062, 0.0063, 0.0064,0.0065, 0.0066, 0.0067, 0.0068, 0.0069, 0.0070, 0.0071, 0.0072, 0.0073,0.0074, 0.0075, 0.0076, 0.0077, 0.0078, 0.0079, 0.0080, 0.0081, 0.0082,0.0083, 0.0084, 0.0085, 0.0086, 0.0087, 0.0088, 0.0089, 0.0090, 0.0091,0.0092, 0.0093, 0.0094, 0.0095, 0.0096, 0.0097, 0.0098, 0.0099, 0.0100,0.0101, 0.0102, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109,0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118,0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127,0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136,0.0137, 0.0138, 0.0139, 0.0140, 0.0141, 0.0142, 0.0143, 0.0144, 0.0145,0.0146, 0.0147, 0.0148, 0.0149, or 0.0150% (w/w) MIT. In someembodiments of systems, systems comprise said compositions and one ormore of a thermocycler, a fluorescence detector, a computer, softwarefor controlling said thermocycler and/or said fluorescence detector, adisplay, a pipettor, sample vessels, tubes configured for holding amaster mix, a refrigerator, storage unit, incubator, and/or a freezer.

The technology finds use in the production of master mixes forcommercial scale manufacturing of reagents for nucleic acidamplification (e.g., PCR (e.g., RT-PCR)) and in the use of nucleic acidamplification by end users (e.g., researchers, clinicians, etc.)

In some embodiments, the technology is useful for PCR. Certain basicprinciples of PCR that may find use in embodiments herein are described,for example, in U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and4,965,188, each of which is incorporated by reference in its entirety.Basic PCR is used to amplify a sample of target DNA for analysis orother uses. PCR uses multiple cycles of denaturation, annealing ofprimer pairs to opposite strands, and primer extension to increase copynumbers of a target nucleic acid sequence exponentially. The basic PCRreaction involves copying the strands of the target DNA and then usingthe copies to generate additional copies in subsequent cycles. Thetemperature of a double-stranded target DNA is elevated to a “denaturingtemperature” (e.g., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91°C., 92° C., 93° C., 94° C., 95° C., 97° C., 98° C., 99° C., or rangestherebetween (e.g., 92-97 ° C.)) to denature the DNA and the temperatureis then reduced to an “annealing temperature” (e.g., 48° C., 50° C., 52°C., 54° C., 56° C., 58° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65°C., 66° C., 67° C., 68° C., 69° C., 70° C., 72° C., 74° C., or rangestherebetween (e.g., 62-72° C.)) to anneal at least one primer to eachstrand of the denatured target DNA. In some embodiments, primers areused as a pair—a forward primer and a reverse primer—and can be referredto as a primer pair or primer set. In some embodiments, the primer setcomprises a 5′ upstream primer that is capable of hybridizing with the5′ end of one strand of the denatured target DNA and a 3′ downstreamprimer that is capable of hybridizing with the 3′ end of the otherstrand of the denatured target DNA. Once a given primer binds to thestrand of the denatured target DNA, the primer is extended by the actionof a polymerase (e.g., at the annealing temperature or at a distinct“extension temperature” (e.g., 65° C., 66° C., 67° C., 68° C., 69° C.,70° C., 71° C., 72° C., 73° C., 74° C., 75° C., or ranges therebetween).In some embodiments, the polymerase is a thermostable DNA polymerase,for example, a Taq polymerase (or suitable variants thereof (e.g.,AMPLITAQ GOLD, CRIMSON TAQ, DEEP VENTR, etc.)). The product ofextension, which sometimes may be referred to as an amplicon, is thendenatured from the resultant strands and the process is repeated. Insome embodiments, the devices and methods provided herein are useful forthe cycling of a nucleic-acid-containing sample through the varioustemperature steps of a PCR reaction. In some embodiments, the PCR is areverse transcriptase polymerase chain reaction (“RT-PCR”), which isknown in the art; see, e.g., Bustin (2000) “Absolute quantification ofmRNA using real-time reverse transcription polymerase chain reactionassays” Journal of Molecular Endocrinology 25: 169-93, incorporatedherein by reference.

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation.

EXAMPLES Example 1

During the development of embodiments of the technology provided herein,experiments were conducted in which master mixes comprising 0, 0.015,0.05, and 0.1% (w/w) PROCLIN 950 (corresponding to 0, 0.0015, 0.005, and0.01% (w/w) MIT concentrations, respectively) were used to prepareRT-PCR reaction mixtures that did not comprise template (2 replicatesper condition). The primers and probes in the reaction mixtures targetHIV-1 nucleic acids. The fluorescent DNA-intercalating dye, EVA Green,was included in each reaction to monitor the accumulation ofnon-specific amplification products. Realtime EVA Green fluorescencesignals from RT-PCR reactions comprising 0, 0.15, 0.05. and 0.1% (w/w)PROCLIN 950 are shown in FIG. 1. Master mixes comprising MIT (fromaddition of PROCLIN 950) exhibited decreased levels of non-specificproduct formation (as demonstrated by delayed EVA Green signals)compared to reactions that did not comprise MIT.

Example 2

During the development of embodiments of the technology provided herein,experiments were conducted to simulate the preparation of bulk RT-PCRreagents by a commercial manufacturing process. In particular, prototypeHIV-1 assay master mixes comprising 0, 0.015, 0.05, and 0.1% (w/w)PROCLIN 950 (corresponding to 0, 0.0015, 0.005, and 0.01% (w/w) MITconcentrations, respectively) were incubated at 2 to 8° C. for 16 hours.RT-PCR was then performed with each master mix in the absence of HIV-1target RNA (2 replicates per condition). The fluorescentDNA-intercalating dye. EVA Green was included in each reaction tomonitor the accumulation of non-specific amplification products.Realtime EVA Green fluorescence signals from RT-PCR reactions comprising0, 0.015, 0.05, and 0.1% (w/w) PROCLIN 950 are shown in FIG. 2. Mastermixes comprising MIT exhibited decreased levels of non-specific productformation (as demonstrated by delayed EVA Green signals) compared toreactions that did not comprise MIT. The data also indicated that thedifference in the Ct value for reactions with MIT and without MIT wasmore pronounced in this experiment compared to the experiment describedin Example 1. Accordingly, the data indicated that MIT and/or PROCLIN950 provided improved stability to the master mix.

Example 3

During the development of embodiments of the technology provided herein,experiments were conducted to test RT-PCR in the presence of MIT in aprototype HIV-1 assay master mix. Prototype HIV-1 assay master mixescomprising 50 copies of HIV-1 target RNA and either 0.015% (w/w) PROCLIN950 (0.0015% (w/w) MIT) or comprising no PROCLIN 950 were tested byRT-PCR (8 replicates each condition). HIV-specific realtime fluorescencesignals from HIV-1 RT-PCR reactions comprising 0 or 0.015% (w/w) PROCLIN950 are shown in FIG. 3. The data indicated that RT-PCR comprisingPROCLIN 950 exhibited increased signal compared to reactions containingno PROLCIN.

Example 4

During the development of embodiments of the technology provided herein,experiments were conducted to test RT-PCR in the presence of MIT in aprototype CT/NG assay master mix. Prototype CT/NG assay master mixescomprising 0.1 inclusion forming units of Chlamydia trachomatis andeither 0.015% (w/w) PROCLIN 950 (0.0015% (w/w) MIT) or comprising noPROCLIN 950 were tested by RT-PCR (8 replicates each condition).CT-specific realtime fluorescence signals from CT/NG RT-PCR reactionscomprising 0 or 0.015% (w/w) PROCLIN 950 (0.0015% (w/w) MIT) are shownin FIG. 4. The data indicated that reactions comprising PROCLIN 950exhibited improved signal compared to reactions containing no PROCLIN950. Further, the prototype CT/NG assay uses different primer and probesets than the prototype HIV-1 assay described in Example 3. Accordingly,the data indicated that the PCR enhancing function of MIT is not limitedto specific primer/probe sequences.

Example 5

During the development of embodiments of the technology provided herein,experiments were conducted to simulate producing a bulk PCR reagent fora HI V-I assay. In particular, master mixes comprising 0, 0.015, 0.05,and 0.1% (w/w) PROCLIN 950 (corresponding to 0, 0.0015, 0.005, and 0.01%(w/w) MIT concentrations, respectively) were incubated at 2 to 8° C. for16 hours. RT-PCR was then performed with each master mix using 50 copiesof HIV-1 RNA (8 replicates per condition). HIV-specific realtimefluorescence signals from HIV-1 RT-PCR reactions comprising 0, 0.015,0.05, and 0.1% (w/w) PROCLIN 950 are shown in FIG. 5. The data indicatedthat master mixes comprising PROCLIN 950 exhibited improved stability(as demonstrated by the increase signal of the RT-PCR curves) comparedto reactions containing no PROCLIN 950.

All publications and patents mentioned in the above specification areherein incorporated by reference in their entirety for all purposes.Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in the artare intended to be within the scope of the following claims.

We claim:
 1. A method of detecting a nucleic acid in a realtime PCRcomposition, said method comprising providing a reaction compositioncomprising a polymerase, a first primer, a second primer, nucleotides,and 0.0001 to 0.01% (w/w) MIT.
 2. The method of claim 1, wherein saidMIT is a PCR enhancer.
 3. The method of claim 1 further comprisingadding a sample to said reaction composition.
 4. The method of claim 1further comprising providing a detectably labeled probe or anintercalating dye.
 5. The method of claim 1 further comprisingthermocycling said reaction composition.
 6. The method of claim 1further comprising measuring a fluorescence emission of said reactioncomposition.
 7. The method of claim 16 further comprising storing arealtime PCR reagent comprising said 0.0001 to 0.01% (w/w) MIT and atleast one of said polymerase, said first primer, said second primer,and/or said nucleotides for at least 1 day.
 8. The method of claim 7comprising storing said realtime PCR reagent for at least 1 week.
 9. Themethod of claim 7 comprising storing said realtime PCR reagent for atleast 1 month.
 10. The method of claim 7 wherein said realtime PCRreagent is used to prepare said reaction composition.
 11. A method ofproducing a realtime PCR reagent comprising mixing a PCR enhancerwherein said PCR enhancer is 0.0001 to 0.01% (w/w) MIT with at least oneof a polymerase, a first primer, a second primer, and/or nucleotides.12. The method of claim 11 wherein said realtime PCR reagent has avolume greater than 1 liter.
 13. The method of claim 12 furthercomprising storing said realtime PCR reagent for at least 1 day.
 14. Themethod of claim 12 further comprising storing said realtime PCR reagentfor at least 1 week.
 15. The method of claim 12 further comprisingstoring said realtime PCR reagent for at least 1 month.
 16. The methodof claim 12 further comprising mixing into said realtime PCR reagent areference dye, a detectably labeled probe, an intercalating dye, a thirdprimer, and/or a fourth primer.